tatematsu, masae citation [journal of …...the samp3 strain, kindly provided by dr. toshio takeda,...

TitleGastric Carcinogenesis and Intestinalization Induced by N-methyl-N-nitrosourea in the Senescence-Accelerated Mouse (SAMP3)(本文(Fulltext) )

Author(s)

YAMAMOTO, Masami; TSUKAMOTO, Tetsuya; SAKAI,Hiroki; HIRATA, Akihiro; YANAI, Tokuma; MASEGI,Toshiaki; HIRANO, Kazuyuki; FURIHATA, Chie;TATEMATSU, Masae

Citation [Journal of Toxicologic Pathology] vol.[16] no.[1] p.[33]-[39]

Issue Date 2003

Rights The Japanese Society of Toxicologic Pathology (JSTP) (日本毒性病理学会)

Version 出版社版 (publisher version) postprint

URL http://hdl.handle.net/20.500.12099/31929

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

J Toxicol Pathol 2003; 16: 33�39

Original

Gastric Carcinogenesis and Intestinalization Induced by N-methyl-N-nitrosourea in the Senescence-Accelerated Mouse (SAMP3)

Masami Yamamoto1, Tetsuya Tsukamoto1, Hiroki Sakai2, Akihiro Hirata2, Tokuma Yanai2, Toshiaki Masegi2, Kazuyuki Hirano3, Chie Furihata4, and Masae Tatematsu1

1Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1�1 Kanokoden, Chikusa-ku, Nagoya 464�8681, Japan

2Department of Veterinary Pathology, Gifu University, 1�1 Yanagido, Gifu 501�1193, Japan3Laboratory of Pharmaceutics, Gifu Pharmaceutical University, 5�6�1 Mitahorahigashi, Gifu 502�0003, Japan4College of Science and Engineering, Aoyama Gakuin University, 1�1 Morinosato-Aoyama, Atsugi, Kanagawa

243�0123, Japan

Abstract: The relationship between gastric neoplasia and intestinal metaplasia, a process whose incidence increaseswith age, remains controversial. In the present experiment, we therefore investigated induction of both intestinalmetaplasia and gastric cancers in the Senescence-accelerated mouse (SAMP3/Aicc) treated with N-methyl-N-nitrosourea(MNU). Seven-week-old animals of both sexes received the carcinogen in their drinking water at 120 or 60 ppm onalternative weeks for 10 weeks, or continuously at 30 ppm for the same period, then maintained without further treatmentuntil sacrificed at week 50. The incidences of adenocarcinomas in the 120, 60, and 30 ppm MNU treated and controlgroups were 6/10 (60%), 5/16 (31.3%), 1/17 (5.9%), and 0/6 (0%) in males, and 2/7 (28.6%), 3/13 (23.1%), 1/6 (16.7%),and 0/9 (0%) in females, respectively. All neoplasms were of well-differentiated type, mainly consisting of gastricepithelial type cells. With immuno- and enzyme-histochemistry, intestinal alkaline phosphatase (I-ALP) positiveintestinal absorptive cell-like elements were observed in regions of hyperplasia, adenomas, and adenocarcinomas ofMNU treated mice, but no phenotypic expression of goblet or Paneth cells was found. In the control mice, gastricmucosa showed no intestinal epithelium phenotype. Since the degree of appearance of I-ALP positive cells in the lesionsin SAMP3/Aicc mice was not different from that found for six other strains mice in our previous work, the resultssuggest that Senescence-acceleration may not influence intestinalization in the gastric mucosa or induction of gastriccarcinomas. (J Toxicol Pathol 2003; 16: 33�39)

Key words: Senescence-Accelerated Mouse (SAM), MNU, gastric cancer, intestinal metaplasia, susceptibility

Introduction

Gastric adenocarcinomas display specific histologicaland biological characteristics and have been classifiedhistologically into two types, intestinal and diffuse1 ordifferentiated and undifferentiated2. Furthermore,cytobiologically, cells of human stomach cancers have beenclassified into a gastric epithelial cell type (consisting ofsurface mucous and pyloric gland cells) and an intestinalepithelial cell type (intestinal absorptive and goblet cells) onthe basis of their phenotypic expression3�5. Intestinal

metaplasia is commonly seen in human gastric mucosa.While it has been reported to be a putative preneoplasticlesion, associated in particular with intestinal type gastriccancers6�8, this conclusion is controversial since early gastrictumors are mainly composed of gastric epithelial cells andthe phenotypic change to intestinal type occurs along withtumor progression9�11.

For analysis of gastric carcinogenesis, experimentalanimal models are very useful. One rat model featuring N-methyl-N�-nitro-N-nitrosoguanidine (MNNG) treatment12,has been widely used which demonstrates the same intestinalphenotypic changes in the stomach in neoplasia as shown inthe human case13. Adenocarcinomas induced by MNNG inthe rat stomach are mainly of differentiated type andinduction of undifferentiated type stomach cancers isrelatively difficult. The mouse also has advantages,especially the recently genetically engineered strains. We

Received: 6 November 2002, Accepted: 21 January 2003Mailing address: Tetsuya Tsukamoto, Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1�1 Kanokoden, Chikusa-ku, Nagoya 464�8681, JapanTEL: 81-52-762-6111 (ext. 7062) FAX: 81-52-763-5233E-mail: [email protected]

34 Gastric Carcinogenesis in the SAMP3

have established mouse models for stomach carcinogenesisusing N-methyl-N-nitrosourea (MNU), with particularsimilarities to human gastric cancers from the viewpoint ofhistopathological variation1 4, poorly differentiatedadenocarcinomas as well as signet-ring cell carcinomasbeing induced. However, there are differences with regardto intestinal change in the stomach between the human andmouse cases. We have examined carcinogenesis with MNUin six strains of mice (BALB/cA, C57BL/6N, CBA/JN,C3H/HeN, DBA/2N, and CD-1) and the typical intestinalmetaplasia and intestinal type gastric tumors which appear inthe rat15,16 were not observed, despite the fact that the lesionspositive for intestinal alkaline phosphatase (I-ALP), amarker of intestinal absorptive cells, could be detected withimmuno- and enzyme-techniques17. Since intestinalmetaplasia is commonly observed in the human stomach ofold individuals, it could be considered as an age-relatedchange. The observed differences between humans andmice might therefore be due to variation in life span andaging processes.

Senescence accelerated mice (SAM) have been derivedfrom AKR/J mice by Takeda and co-workers18 and SAMP1,P2, P3, P6, P7, P8, P9, and P10 strains (P for prone) havenow been established19. They have a normal developmentbut exhibit accelerated senescence. They thus showmoderate to severe loss of activity, deterioration of hairquality and fallout, skin coarseness, periophthalmic lesions,increased lordokyphosis of the spine, and a shortened lifespan. Each SAMP strain has a strain-specific pathologicalphenotype, for example, deficits in learning and memorybeing found in SAMP8 and P10, senile amyloidosis inSAMP1, P2, P7, and degenerative joint disease oftemporomandibular joints in SAMP3. SAMP3 has a lifespan of approximately 16 months, while the other strains liveon average for 10�13 months. Since induction of intestinal

metaplasia and gastric tumors generally requires a relativelylong experimental period the SAMP3/Aicc (subline ofSAMP3 at Aichi Cancer Center) strain was selected for thepresent investigation of the relationship between intestinalchange and aging in gastric mucosa as well as gastrictumorigenesis due to MNU.

Materials and Methods

Experimental design The SAMP3 strain, kindly provided by Dr. Toshio

Takeda, Kyoto University, Kyoto, Japan, was maintained inthe Animal Facility of Aichi Cancer Center ResearchInstitute with brother-sister mating for more than 15generations. The substrain is named SAMP3/Aicc. Allanimals were housed in plastic cages with hard wood chipsin an air-conditioned room with a 12 h light-12 h dark cycleand received basal diet (Oriental NMF, Oriental Yeast Co.,Tokyo, Japan) ad libitum. With administration of the gastriccarcinogen, MNU (Sigma Chemical Co., St Louis, MO), at120 ppm in the drinking water continuously for 18 weeks,six other strains of mice (BALB/cA, C57BL/6N, CBA/JN,C3H/HeN, DBA/2N, and CD-1) survived for one year in ourprevious study17. However, a preliminary experimentrevealed that the same administration of MNU caused earlymortality of SAMP3/Aicc mice. Thus MNU was given onalternate weeks at 120 and 60 ppm or continuously at 30ppm, as shown in Fig. 1. The carcinogen solutions werefreshly prepared three times per week and administered inlight-shielded bottles. Controls received tap water. Theanimals were sacrificed at week 50 and necropsies were alsoperformed on all those that died or were killed uponbecoming moribund. The excised stomachs were fixed in1% acetic acid in 95% ethanol, cut into about 6 strips, andembedded in paraffin.

Fig. 1. Experimental design. Seven-week-old male mice were administered MNU at 120 ppm (black), 60ppm (bold hatching) or 30 ppm (light hatching) following the indicated schedules or water (open).S; sacrifice.

Yamamoto, Tsukamoto, Sakai et al. 35

Histopathological analysesAll tissue sections were stained with hematoxylin and

eosin (H&E). Neoplastic lesions of glandular stomach wereclassified into adenomas and adenocarcinomas according tothe criteria previously reported14. Tissue samples withoutgood preservation or without sufficient amounts were notfurther analyzed.

Immunohistochemical, enzyme histochemical andmucin histochemical analyses

Anti-rat Pg120 and anti-rat I-ALP antibodies wereprepared16 for immunohistochemical analyses. The avidin-biotin complex method with a Vectastain ABC kit (VectorLaboratories Inc., Burlingame, CA) was used for detection.I-ALP enzyme activity was demonstrated using nitro bluetetrazolium chloride/5-bromo-4-chloro-3-indoxyl phosphate(NBT/BCIP) (Roche Diagnostics, Tokyo, Japan) in thereaction mixture containing 1 mM of levamisole. For mucinhistochemistry, Alcian blue (pH 2.5) periodic acid Schiff(AB-PAS) and paradoxical concanavalin A staining (PCS)methods21 were employed.

When a lesion contained I-ALP positive epithelial cells,it was judged as IAP-positive. Because the cells in thenormal pyloric mucosa just adjacent to the duodenumexhibited a mixture of gastric and intestinal phenotypes, thisarea was excluded from the analysis.

Statistical analysisThe incidences of stomach tumors and I-ALP positive

lesions were analyzed using the one-tailed Fisher�s exactprobability test.

Results

Histopathological findings for tumorsStomach cancers induced by MNU were all well-

differentiated adenocarciomas. The degree of invasion wasup to the submucosa, with no involvement of the subserosao r s e r o s a . F ig . 2 s h o w s a we l l - d i f f e r e n t i a t edadenocarcinoma invading the submucosa. The incidences ofhyperplasias, adenomas, and adenocarcinomas in theglandular stomach are summarized in Table 1. Most of thelesions developed in the pyloric mucosa. Hyperplasias wereobserved in most animals treated with MNU, whereasincidences of adenomas and adenocarcinomas were dose-dependent. There were no significant differences inincidence between males and females.

Cellular phenotypes of neoplastic lesionsIn the normal pyloric mucosa, Pg1 and PCS were

clearly positive in pyloric gland cells. In all hyperplasias,Pg1 staining became weak or negative, while PCS stainingwas still positive. In tumors, Pg1 was very low or negativebut PCS expression varied from region to region. Staining ofPg1 and PCS was decreased along with the degree ofhistological evidence of malignancy. Pg1 decreased firstfollowed by PCS.

No typical intestinal metaplastic lesions, showinggoblet or complete differentiated intestinal absorptive celltypes were observed. Immunohistochemical and enzymehistochemical analyses revealed the existence of I-ALPpositive areas with incomplete intestinal absorptive cells inhyperplasias (Fig. 3) and tumors. I-ALP was found in thecovering epithelium associated with a change of mucin typefrom PAS positive to negative or weakly Alcian bluepositive. The incidences of I-ALP positive lesions in theMNU treated and in control groups are summarized in Table2. Adenocarcinomas tended to have lower I-ALP positivitythan adenomas. In control animals, the stomach mucosa didnot exhibit any intestinal features.

Discussion

It is clear that SAMP3/Aicc mice have highersusceptibi li ty to MNU carcinogenicity than othercommercially available mice and administration of 120 ppmcontinuously for 18 weeks caused tumors in multiple organsbefore stomach lesions developed. Thus milder treatmentschedules were selected here. We earlier reported that theproportion of undifferentiated adenocarcinomas in theglandular stomach induced by MNU depends on theconcentration of the carcinogen22 and in this study, alladenocarcinomas were of well-differentiated type.

We have described intestinalization observed in gastriccarcinogenesis in six strains of mice (BALB/cA, C57BL/6N,CBA/JN, C3H/HeN, DBA/2N, and CD-1) induced by MNUto be limited to ectopic I-ALP expression withouthistological change17. The percentage of I-ALP positivecells in areas of hyperplasia increased from 10 to 52 weeks inall six strains. At 50 weeks in our SAMP3 strain, the valueswere not different from those at 52 weeks in the other strains(63.6�95.8%), so that senescence does not appear toaccelerate intestinalization in the gastric mucosa.

All lesions observed here were heterogeneous for I-ALP expression, and none consisted only of I-ALP positivecells, in line with our findings for the other six mice strains17.Since adenomas and carcinomas are thought to bemonoclonal lesions, this random expression must be theresult of heterozygosity. In man, gastric and intestinal mixedtypes in single crypts are observed in intestinal metaplasia23.Whether due to alteration in differentiation pathways ormutations, the heterogeneity indicates that intestinalizationis not necessary for carcinogenesis in the mouse gastricmucosa.

Recently, not only aging but Helicobacter pylori (H.pylori) infection has been thought to be a major risk factorassociated with intestinal metaplasia in man24,25. In theimmunocompetent mouse, infection with H. pylori isdifficult to establish and histological gastritis has not beenshown26. However, Helicobacter felis infection in micecauses chronic atrophic gastritis and slight intestinalmetaplasia, featuring Alcian blue positive mucous cells butno clear intestinal type goblet cells27,28. Mongolian gerbilsare also sensitive to H. pylori29, with induction of chronic


Fig. 2. A well-differentiated adenocarcinoma in a 120 ppm MNU-treated SAMP3/Aicc mouse,invading the submucosa. H&E staining. × 30.

Table 1. Incidences of Neoplastic Lesions in SAMP3 Mice Treated with MNU (%)

HistologyHyperplasia (%) Adenoma (%) Adenocarcinoma (%)

Male 120 11 11 (100)a) 10 (90.9) a) 6 (54.5) b), c)

60 16 15 (93.8)a) 13 (81.3) a) 5 (31.3)30 17 17 (100) 10 (58.8) b) 1 (5.9)

0 6 0 0 0Female 120 7 7 (100)a) 6 (85.7) a) 2 (28.6)

60 13 12 (92.3)a) 9 (69.2) a) 3 (23.1)30 6 5 (83.3)a) 2 (33.3) 1 (16.7)

0 9 0 0 0

with Fisher�s exact test vs 0 ppm a): p<0.01 b): p<0.05, vs 30 ppm c): p<0.01.

Table 2. Incidences of I-ALP Positive Lesions in the Glandular Stomach in SAMP3 Mice Treated with MNU

Hyperplasia (%) Adenoma (%) Adenocarcinoma (%)MNU (ppm) No. of micea) Total no. I-ALP Total no. I-ALP Total no. I-ALP

of lesionsa) positiveb) of lesionsa) positiveb) of lesionsa) positiveb)

Male 120 8 8 6 (75.0) 14 7 (50.0) 5 0 (0)60 15 14 10 (71.4)c) 17 12 (70.6)c) 5 3 (60.0)30 13 13 8 (61.5) 13 7 (53.8) 1 0 (0)0 4 0 0 0 0 0 0

Female 120 7 7 6 (85.7)c) 9 7 (77.8)c) 1 0 (0)60 10 9 6 (66.7)c) 8 5 (62.5) 2 1 (50.0)30 6 5 4 (80.0)c) 3 2 (66.7) 0 0 (0)0 5 4 0 0 0 0 0

a): Total No. of mice/lesions examined for I-ALP analysis, b): Numbers (%) of lesions containing I-ALP positive cell.with Fisher�s exact test vs 0 ppm c): p<0.05.

MNU (ppm) No. of mice


Fig. 3. An area of hyperplasia in a 120 ppm MNU-treated SAMP3/Aicc mouse. × 80 (A) H&E. (B) I-ALP immunostaining. (C) AB-PAS.


active gastritis from the fundic to the pyloric glands of thestomach. The carcinogens MNU and MNNG do not induceintestinal metaplasia that could be recognized by phenotype,but a combination of carcinogen and H. pylori or H. pylorialone was found to result in intestinal metaplasia with Alcianblue positive clear goblet cells in the gerbil glandularstomach30,31. Chronic gastritis induced by germs may haveinfluence on intestinal metaplasia of gastric epithelium.Using SAM mouse, there is one experiment, entericinflammation develops in SAMP1/Yit, a subline of theSAMP1 mouse, under conventional but not in germ freeconditions32. In our experiment, SAMP3 under conventionalcondition have no significant histopathological change.

Watanabe et al.33 showed that gastric pH influencesintestinal metaplasia in X-irradiated rats. They reported thatthe numbers of intestinal metaplastic lesions, enzymehistochemically ALP positive foci, and areas of type Bmetaplasia (featuring intestinal crypts without Paneth cells),were decreased in the low pH groups with induction ofgastric acid secretion. It is possible that pH similarly affectsintestinal metaplasia in the Mongolian gerbil or mouseinfected with Helicobacter. In our mouse model of gastrictumors with MNU, however, apparent intestinal metaplasiaconsisting of goblet cells or Paneth cells were not inducedprobably because stomach pH might not be affected so muchsince fundic gland cells were well preserved as the lesionsoccur mainly in areas occupied by pyloric glands.

Regarding controls not given carcinogen, there was noremarkable difference between SAMP3/Aicc and othercommercially available mice in terms of I-ALP expression.Thus MNU rather than accelerated aging influencedintestinal metaplasia in gastric mucosa, I-ALP positive areasonly being observed in the carcinogen treated groups.However, they were distributed apparently at random, withno spatial relationship to glandular stomach cancers. Therewas no relation between intestinal metaplasia as shown withI-ALP expression and carcinogenesis. However, chemical,physical, or inflammatory injury such as X-irradiation,carcinogen exposure, or bacterial infection and subsequentregeneration of mucosa may influence the intestinalization.

In conclusion the present study revealed that whereasSAMP3/Aicc mice have a high sensitivity to MNU, theirSenescence-acceleration does not appear to influenceintestinalization of the gastric mucosa and there was no clearrelationship between intestinalization and gastric tumors.

Acknowledgements: This study was supported in part byGrants-in-Aid for Cancer Research from the Ministry ofEducation, Science Sports, Culture and Technology, Japan;by the Ministry of Health, Labour and Welfare of Japan; andby a Grant-in-Aid from the Ministry of Health, Labour andWelfare for a Comprehensive 10 Year Strategy for CancerControl, Japan. We also gratefully thank Dr. Atsushi Abefor expert technical assistance.

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